Otitis media (OM) is one of the most common of childhood diseases and a major public health problem. The disease can lead to middle ear and inner ear pathology and hearing loss. There are fundamental gaps in our understanding of how bacterial pathogens in OM cause the inflammatory response in the middle ear, the mechanism of middle and inner ear interaction, inner ear pathology, and hearing loss. This prompted us to develop a cogent investigation of the molecular processes underlying migration of infectious agents from the middle to the inner ear and the effects of such migration on the ear pathology. Widespread use of antibiotics has resulted in an increase of antibiotic-resistant Streptococcus pneumoniae and an increased potential for chronic OM development and its complications. Although polysaccharide vaccines have been developed, there are more than 90 distinct serotypes for S. pneumoniae, and antibodies against one serotype generally do not protect against another. The current 7-valent pneumococcal conjugate vaccine PCV7 is 100% efficacious against invasive pneumococcal disease of vaccine serotypes in children < 2-years old, but only 34% against acute OM. A protein-based vaccine may overcome this limitation by including antigens conserved among different pneumococcal serotypes. Our long-term goal is to develop novel approaches for prevention and treatment of OM and its complications. In this application, we propose a new strategy for prevention of OM and its complications that relies on a combination of PspA and other pneumococcal proteins. We will utilize different mutant bacterial strains and immunization of animals against these proteins. Our objective is to understand the role of bacterial virulence factors in bacterial invasion, colonization, and pathogenesis in the middle and inner ears for therapeutic targeting and to develop a vaccine composition that optimally neutralizes the most critical protein virulence factors. Our central hypothesis is that PspA and other bacterial proteins affect the pathology of the middle and inner ears. Bacterial components and inflammatory mediators produced in the middle ear pass through the round window membrane into the inner ear, damage cochlear structures, resulting in hearing loss. Our rationale is that knowing how bacterial components affect the pathology of the middle and inner ears will make it possible to design new approaches for the prevention and treatment of OM. These considerations have led to the formulation of our Specific Aims: 1) To identify the role of pneumococcal PspA protein and its combination with other potential pneumococcal vaccine proteins on the middle and inner ear and strategies for prevention of OM; 2) To identify mechanisms of inner ear damage and auditory dysfunction caused by live S. pneumoniae. The results gained from these studies will provide new insights into the roles of S. pneumoniae virulence factors in the development of OM and inner ear complications. To achieve our goal we have assembled a group of PIs who have collaborated in recent years, and who are uniquely qualified to address this challenge with their combined expertise. PUBLIC HEALTH RELEVANCE: These studies will use innovative approaches to determine the effects of important pneumococcal virulence proteins and their combination on the middle and inner ears. Understanding the role of bacterial virulence factors and host defense using mutant strains and immunization experiments may have an important positive impact to facilitate the development of new strategies for prevention and treatment of otitis media and its complications, especially significant in the era of multi-drug resistant bacteria, by selection of bacterial proteins for future vaccine design against pneumococcal otitis media, such as protein- based or conjugate polysaccharide vaccine with pneumococcal proteins as carriers.